The metabolism of lipids, particularly cholesterol, involves the interaction of a number of lipoproteins and apolipoproteins. Very low density lipoprotein (VLDL) and apolipoprotein E (apoE) are key precursor molecules in the production of low density lipoprotein (LDL) and in the overall metabolism of lipids, including cholesterol. LDL is the major cholesterol-transport lipoprotein in human plasma. The VLDL/apoE receptors are expressed in heart, skeletal muscle, and adipose tissue [F. M. Wittmaack et al, Endocrinol., 136(1):340-348 (1995)] with lower levels of expression in the kidney, placenta, pancreas, and brain. This receptor has been suggested to play a role in the uptake of triglyceride-rich lipoprotein particles by specific organs. The cDNA encoding the putative human VLDL receptor was recently cloned [M. E. Gafvels et al, Som. Cell Mol. Genet., 19:557-569 (1993), incorporated by reference herein]. The receptor for LDL is located in coated pits on the surfaces of cells in the liver and other organs.
As depicted in FIG. 1A, in a normal healthy human, the molecules apolipoprotein B48 (Apo-B48), apolipoprotein C-II (Apo-C-II) and Apo E form a chylomicron particle in plasma passing through the intestines, which interacts with a chylomicron remnant receptor in the liver. After metabolism of the chylomicrons taken up by the remnant receptor, the liver produces the primary lipoprotein, VLDL, which contains Apo-E, Apo-C-II and apolipoprotein B100 (Apo B100). VLDL is metabolized into LDL, which binds to the LDL receptor in the liver via Apo B100. The LDL receptor in the liver facilitates the uptake of LDL by receptor-mediated endocytosis. LDL is degraded in lysosomes, and its cholesterol is released for metabolic use.
Defects in the metabolism of such lipoproteins and/or receptors result in several serious metabolic disorders. The human disease familial hyper-cholesterolemia (FH) is caused primarily by one or more mutations in the gene encoding the LDL receptor. FH is characterized clinically by (1) an elevated concentration of LDL; (2) deposition of LDL-derived cholesterol in tendons and skin (xanthomas) and in arteries (atheromas); and (3) inheritance as an autosomal dominant trait with a gene dosage effect. Individuals with FH develop premature coronary heart disease, usually in childhood. Heterozygotes number about 1 in 500 persons, placing FH among the most common inborn errors of metabolism. Heterozygotes have twofold elevations in plasma cholesterol (350 to 550 mg/dl) from birth and tend to develop tendon xanthomas and coronary atherosclerosis after age 20. Homozygotes number 1 in 1 million persons and are characterized by severe hypercholesterolemia (650 to 1000 mg/dl), cutaneous xanthomas which appear within the first 4 years of life, and coronary heart disease which begins in childhood and frequently causes death before age 20. [J. Goldstein et al, “Familial Hypercholesterolemia”, Chapter 48, in The Metabolic Basis of Inherited Disease, 6th ed., C. R. Scrivers et al (eds), McGraw-Hill Information Services Co., NY, N.Y., (1989) pp. 1215-1250].
Another metabolic disorder is familial combined hyperlipidemia (FCH) which was first associated with hyperlipidemia in survivors of myocardial infarction and their relatives. FCH patients generally have one of three phenotypes: (1) elevated levels of VLDL, (2) elevated levels of LDL, or (3) increases in the levels of both lipoproteins in plasma. Unlike FH, FCH appears in only 10 to 20 percent of patients in childhood, usually in the form of hypertriglyceridemia. Homozygosity for the trait may result in severe hypertriglyceridemia. [J. Goldstein et al, “Disorders of the Biogenesis and Secretion of Lipoproteins”, Chapter 44B in The Metabolic Basis of Inherited Disease, 6th ed., C. R. Scrivers et al (eds), McGraw-Hill Information Services Co., NY, N.Y., (1989) pp. 1155-1156]. This disorder is also associated with the appearance of glucose intolerance and obesity in a number of individuals.
The most striking abnormality of FCH is marked elevation of VLDL content of plasma. Increased production of VLDL leads to an expanded plasma pool of VLDL in some individuals, but in others with more efficient lipolysis, it results in increased levels of LDL. FCH is characterized by an excess production of LDL, rather than a genetic defect in the LDL receptor. The LDL receptors of cultured fibroblasts appear to be normal in FCH patients.
Clinical experience suggests that FCH is at least five times as prevalent as FH, occurring in about 1 percent of the North American population. The predilection toward coronary artery disease among patients with this disorder makes it the most prominent known metabolic cause of premature atherosclerosis [J. Goldstein et al, cited above].
When LDL receptors are deficient as in FH (see FIG. 1B), or excess LDL is produced due to excess VLDL as in FCH, the efficient removal of LDL from plasma by the liver declines, and the level of LDL rises in inverse proportion to the receptor number. The excess plasma LDL is deposited in connective tissues and in scavenger cells, resulting in the symptoms of either disorder.
Presently, treatment for FH and FCH is directed at lowering the plasma level of LDL by the administration of drugs, i.e., combined administration of a bile acid-binding resin and an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase for treatment of FH and niacin for treatment of FCH. However, FH homozygotes with two nonfunctional genes are resistant to drugs that work by stimulating LDL receptors. Similarly, such drugs are not particularly effective in FCH. In FH homozygotes, plasma LDL levels can be lowered only by physical or surgical means.
Administration of normal LDL receptor genes by gene therapy using an adenovirus vector has been contemplated for the treatment of FH. Adenovirus vectors are capable of providing extremely high levels of transgene delivery to virtually all cell types, regardless of the mitotic state. The efficacy of this system in delivering a therapeutic transgene in vivo that complements a genetic imbalance has been demonstrated in animal models of various disorders [K. F. Kozarsky et al, Somatic Cell Mol. Genet., 19:449-458 (1993) (“Kozarsky I”); K. F. Kozarsky et al, J. Biol. Chem., 269:13695-13702 (1994) (“Kozarsky II); Y. Watanabe, Atherosclerosis, 36:261-268 (1986); K. Tanzawa et al, FEBS Letters, 118(1):81-84 (1980); J. L. Golasten et al, New Engl. J. Med., 309:288-296 (1983); S. Ishibashi et al, J. Clin. Invest., 92:883-893 (1993); and S. Ishibashi et al, J. Clin. Invest., 93:1885-1893 (1994)]. The use of adenovirus vectors in the transduction of genes into hepatocytes in vivo has previously been demonstrated in rodents and rabbits [see, e.g., Kozarsky II, cited above, and S. Ishibashi et al, J. Clin. Invest., 92:883-893 (1993)].
Recent research has shown that introduction of a recombinant adenovirus encoding the human LDL receptor (“LDLR”) cDNA into the livers of LDL receptor-deficient Watanabe heritable hyperlipidemic (WHHL) rabbits, which mimic the condition of FH, resulted in large, transient reductions in plasma cholesterol. The transient nature of the effect of recombinant adenoviruses in most situations is attributed to the development of cellular immune responses to the virus-infected cells and their subsequent elimination. Antigenic targets for immune mediated clearance are viral proteins expressed from the recombinant viral genome and/or the product of the transgene, which in this case, is the LDL receptor protein [Y. Yang et al, Proc. Natl. Acad. Sci., USA, 91:4407-4411 (May 1994); Y. Yang et al, Immun., 1:433-442 (August 1994)].
Additionally, repeated reinfusions of the LDLR gene-containing adenovirus did not produce similar, subsequent cholesterol reductions due to the development of neutralizing anti-adenovirus antibodies [Kozarsky I and Kozarsky II, cited above; see also Y. Yang et al, Immun., 1:433-442 (August 1994), all incorporated by reference herein].
There remains a need in the art for therapeutic compositions and gene therapy strategies which enable effective treatment and/or prevention of FH and FCH, as well as other defects in lipoprotein metabolism.